Summary: Given the emerging difficulties with malaria drug resistance and vector control, as well as the persistent lack of an effective vaccine, new malaria vaccine development strategies are needed. We used a novel methodology to synthesize and fully characterize multiple antigen peptide conjugates (MAPs) containing protective epitopes from Plasmodium falciparum and evaluated their immunogenicity in four different strains of mice. A di-epitope MAP (T3-T1) containing two T-cell epitopes of liver stage antigen-1 (LSA-1), a di-epitope MAP containing T-cell epitopes from LSA-1 and from merozoite surface protein-1 (MSP-1), and a tri-epitope MAP (T3-CS-T1) containing T3-T1 and a potent B-cell epitope from the circumsporozoite protein (CSP) central repeat region were tested in this study. Mice of all four strains produced peptide specific antibodies, however the magnitude of the humoral response showed marked genetic variation between the different strains of mice. Anti-MAP antibodies recognized stage-specific proteins on the malaria parasites in an immunofluoresence assay (IFA). In addition, serum from hybrid BALB/cJ x A/J CAF1 mice that had been immunized with the tri-epitope MAP T3-CS-T1 successfully inhibited the malaria sporozoite invasion of hepatoma cells in vitro. Spleen cells from immunized mice also showed a genetically restricted cellular immune response when stimulated with the immunogen in vitro. This study indicates that well-characterized MAPs combining solid phase synthesis and conjugation chemistries are potent immunogens and that this approach can be utilized for the development of subunit vaccines.